F23 Lab 7 Report

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University of Texas, Dallas *

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3380

Subject

Biology

Date

Dec 6, 2023

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docx

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4

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BIOL 3380 Experiment 7 - Lab Report Western Blot Analysis of rGFP Fractions Name: Rania Mohamed Circle your lab section: 005 T–AM W-AM R-AM F-AM T–PM W-PM R-PM F-PM Instructor: Mazambani Simarashe Graduate TA: Falah Rafah Date: 11/15/2023 Partner: Varshini 7- 1
Experiment 7 – Lab Report Western Blot Analysis of rGFP fractions Simply edit this word document by inserting your answers after each question/space. Please denote your typed answers by using bold , italic s, or colored font . Do NOT renumber/reorganize the questions. “Showing your work/calculations” can be achieved by typing it out, pasting in a clear image of your handwritten work, or using handwritten annotation on the document (if you have the technology to do so). 1. (10pts) You will receive these points if you performed the SDS-PAGE and the Western Transfer correctly. 2. (2pts) What is the GENERAL purpose of a Western Blot? Detect presence of rGFP by using a specific antibody 3. (2pts) Ponceau S is a red dye that reversibly binds to all proteins. We used this dye to stain the membrane prior to developing the Western blot with antibodies. One student’s blot did not have any bands after performing the Ponceau S stain. Describe one likely procedural error during the transfer set-up that would account for this observation. The protein side of the membrane was facing down instead of upside and so the dye did not bind to it 4. (2pts) Why did we use a primary antibody that recognizes the Xpress epitope tag and not the His 6 tag to detect rGFP? The his6 tag could have bound other contaminants besides rGFP but xpress epitope tag is more specific for the antibody 5. (5pts) Make a figure of your Western blot result with appropriate titles and labels. Please see page 7-4 6. (2pts) Describe the data from the G0, G3, and GCE lanes of your Western blot result. Are those results expected? Explain why or why not. G0 had no bands. G3 and GCE lanes had bands in them. These results were expected because at G0 there was no IPTG while G3 was induced with IPTG and rGFP was expressed 7. Based on your knowledge of Experiments 3-7, when you developed the Western blot: a. (1pt) What MW band in your elution lanes are you expecting to observe in the final result? About 32 kD 7- 2
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